TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.5 DETECTION/Molecular Methods

Section 2.5.5
Non-Radioactive
PSTVd Detection Using
Biotin-Labeled Probes

The non-radioactive PSTVd detection system is a modification of the

conventional radioactive Nucleic Acid Spot Hybridization test (Rad-
NASH). The Rad-NASH test is based on the hybridization of a highly
radioactive PSTVd complementary nucleic acid with the PSTVd bound to
the nitrocellulose membrane, which allows autoradiographic detection of
the hybrid.

In the non-radioactive method, a biotinylated DNA-probe is used. The

hybridization is observed visually on the membrane using an enzymatic
reaction.

The test can be completed in a laboratory with minimum facilities. In this

test, a very small amount of the test sample is spotted onto the
membrane. Then, the areas not utilized by the sample are blocked, and
 the samples are hybridized with the PSTVd probe. In the last step, the
biotinylated probe is reacted with a specific enzyme-labeled conjugate,
and the complex is detected by means of an appropriate substrate.

1. Labeling System (Nick-Translation)

The most appropriate method for the preparation of biotinylated probes is

“nick-translation." This method consists of creating free 3’OH groups in
the double-stranded DNA molecule (the plasmid contains the cDNA of
the viroid or the virus to be detected). These free 3’OH groups are then
used as initiation points for new DNA synthesis by the enzyme DNA
polymerase, which incorporates nucleotides present in the solution. In
this way, labeled nucleotides present in the reaction mixture are
incorporated into the new DNA molecule.

For the labeling reaction, use the BRL Nick Translation Reagent Kit (cat.
No. 8160 SB) and a biotinylated nucleotide (e.g., biotin-7-dATP BRL cat.
No. 9509 SA or biotin-14-dATP BRL cat. No. 9524 SA). Store at –20°C.
Mix the following in an Eppendorf tube:

Distilled H20 … µl *.*

Sol. A (all the dNTPs except the
one labeled form) 5 µl
Plasmid (1µg) … µl *
biotinylated nucleotide (biotin-
dATP sol. conc. 0.4 mM) 2.5 µl

* Volume depends on plasmid concentration.

**Up to 45 µl.

Mix and add 5 µl of sol. C (Polymerase I/DNase I).

Centrifuge briefly.

Incubate at 15°C for 90 minutes.

After incubation add:

Distilled H20 3 µl
Sol. D (stop-buffer) 5 µl
SDS 5% 1 µl
Yeast t-RNA (20 mg/ml stock sol.) 1 µl
NH4 –Ac 7.5M 30 µl

Mix and add 225 µl Ethanol. Precipitate at –20°C overnight or at –70°C

for 1 hour.

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 Centrifuge for 15 minutes, eliminate the supernatant, and add the
following to the pellet:

1 X SSC 90 µl
Na-Ac 3 M pH 5.2 10 µl

Mix carefully and add 225 µl Ethanol.

Precipitate at – 20°C and centrifuge for 15 minutes.

Eliminate the supernatant and dry the pellet. Then

re-suspend in 100 µl 1XSSC.

Store at – 20°C until use.

2. Control Testing

The control testing is performed to estimate the efficiency of the

biotinylated nucleotide incorporation by determining the quality of the
DNA preparation. With a good incorporation, it is possible to visualize
around 1 pg of biotin-labeled DNA.

Prepare four serial 1:10 dilutions (2 µl biotinylated DNA + 18 µl water:

1:10, 1:100, 1:1,000, and 1:10,000).

Spot 5 µl of the pure DNA preparation and 5 µl of each of the four

dilutions onto a nitrocellulose membrane, and let dry at room
temperature.

To complete the control testing of the incorporation, start from “Blocking

and Visualization.”

A good incorporation is reached when the visualization of the color

reaction is possible to the fourth spot.

3. Prehybridization

Put the membrane in the hybridization plastic bag, seal, and cut a corner
of the bag.

Just before use, denature the Herring Sperm DNA by heating at 100°C
for 10 minutes (in a water bath). Chill on ice immediately.

Add the prehybridization buffer to the bag. Mix well and seal the bag,
avoiding air-bubbles. Incubate at 42°C in a water bath for 2– 4 hours.

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 Prehybridization buffer: (10ml)
Stock sol. Final Conc. Amount
Formamide 50% 5 ml
20X SSC 5X 2.5 ml
Denhardt’s sol. 50X 5X 1 ml
Sodium phosphate 1M pH 6.5 25 mM 250 µl
H20 0.75 ml
*Herring Sperm DNA 0.5 mg/ml 0.5 ml
(10 mg/ml)
*Denature immediately before use and chill on ice; then add to other components.

4. Hybridization

Hybridization buffer: (10ml)

Stock sol. Final Conc. Amount
Formamide 45% 4.5 ml
20X SSC 5X 2.5 ml
Denhardt’s sol. 50X 1X 200 µl
Sodium phosphate 1M pH 6.5 20 mM 200 µl
Dextran sulfate 50% 5% 1 ml
H20 1.3 ml
*Herring Sperm DNA 0.2 mg/ml 200 µl
(10 mg/ml)*
*Denature immediately before use and chill on ice, then add to the other components.

Remove the bag containing the membrane from the water bath. Cut out a
corner of the plastic bag and remove the prehybridization buffer.

Denature the Herring Sperm DNA and the probe in a water bath at 100°C
for 10 minutes, and chill on ice immediately.

Add the hybridization buffer to the bag. Mix well and seal, avoiding air-
bubbles.

Incubate overnight in a water bath at 42°C.

The next day, remove the bag, cut out a corner, and remove the
hybridization buffer (save and store it at – 20°C; it can be reused).

5. Washing

Washing steps are done in a plastic tray using a rotary shaker. See the
following table for buffers and conditions. Pre-heat buffer N° 3 to 50°C.

6. Blocking and visualization

See the following table for buffers and conditions. Pre-heat buffer N° 5 to
60°C. Dissolve NBT and BCIP independently in 100 µl of n, n-
dimethylformamide (DMF) and add to the buffer in a container covered
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 with aluminum foil. The color-development reaction must also be
performed in a covered tray.

Caution: DMF solution is highly toxic and is absorbed by the skin. Wear
gloves when preparing the NBT/BCIP substrate solution.

7. Preparation of buffers and solutions required for the non-

radioactive NASH

a) 20XSSC: 1l
NaCl (3M) 175.32 g
Na3 citrate 2H2O (0.3 M) 88.23 g
Dissolve in 800 ml of distilled water. Adjust pH to 7.0 with a few
drops of a 10-N solution of NaOH. Complete the volume to 1 l.
Store at 4°C.

b) Denhardt’s Solution 50X:

Ficoll 1%
Polyvinylpyrrolidone (PVP) 1%
BSA (Fraction V) 1%
Dissolve in distilled water. Filter through a disposable Nalgene
filter. Dispense into 25-ml aliquots and store at –20°C.

c) Herring Sperm DNA: 10 mg/ml

Dissolve 1 g of herring sperm DNA in 100 ml of sterile distilled
water at 4°C overnight (with magnetic stirrer).
At room temperature, pass 4 times through a hypodermic syringe
strongly to break the DNA. Incubate in water bath at 100°C for 10
minutes and cool on ice. Divide into aliquots and store in sterile
tubes at –20°C.

d) Deionized formamide:
Mix 200 ml of formamide and 7 g of mixed-bed, ion-exchange
resin (Bio-Rad AG 501-X8, 20– 50 mesh) in a beaker. Stir for 1–
2 hours at room temperature. Filter through Whatman No.1 filter
paper. Repeat the operation with 7 g of resin and filter again.
Divide into aliquots and store at –20°C.

e) SDS: 20%
Dissolve 20 g of electrophoresis-grade SDS in 90 ml of distilled
water. Heat to 68°C to assist dissolution. Adjust pH to 7.2 by
adding a few drops of concentrated HCl. Adjust volume to 100
ml. Wear a mask when weighing SDS. There is no need to
sterilize SDS 20%. Store at room temperature.

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Buffer Time Temp. Amount Prep. Final concentration Stock ml

Washing:
N° 1 2x5’ Room-T 250 ml 500 ml SSC 2X 20X 50 ml
SDS 0.1% 20% 2.5 ml

N° 2 2x5’ Room-T 250 ml 500 ml SSC 0.2X 20X 5 ml

SDS 0.1% 20% 2.5 ml

N° 3 2x15” 50°C 250 ml 500 ml SSC 0.16X 20X 4 ml

SDS 0.1% 20% 2.5 ml

N° 4 3” Room-T 200 m 200 ml SSC 2X 20X 20 ml

Blocking:

N° 5 600 ml Tris-HCl 2M 30 ml
0.1 M pH 7.5
NaCl 0.1 M 5M 15 ml

N° 6 60– 90 min 60°C 100 ml 100 ml Buffer N° 5 3g

BSA 3%

N° 7 20’ Room-T 10 ml Buffer N° 5

SA-AP 1:1000 10 µl

N° 8 2x15’ Room-T 250 ml Buffer N° 5

N° 9 10’ Room-T 200 ml 200 ml Tris-HCl 0.1M 1M 20 ml

pH 9.5
NaCl 0.1 M 5M 4 ml
MgCl2 0.05 M 1M 10 ml

N° 10 30’ – 2hs Room-T 30 ml Buffer N° 9

NBT 6 mg
BCIP 3 mg

N° 11 2x5” Room-T 200 ml Distilled water

f) Yeast transfer-RNA: 20 mg/ml

Weigh 0.25 g of t-RNA and resuspend it in 10 ml of sterile
distilled water. Add 5 ml of phenol (1/2 vol.) and 5 ml of
chloroform (1/2 vol.). Mix well with vortex and centrifuge to
separate phases. Collect the supernatant and repeat extraction
with phenol and chloroform two more times.

Extract once with 10 ml of chloroform and transfer supernatant to

a clean tube.

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 Add 1 ml of Na-acetate 3 M pH 5.2 (1/10 vol.) and 25 ml of
ethanol (2.5 vol.)

Precipitate overnight at –20°C; then centrifuge at 10,000 rpm for

15 minutes. Dry the pellet under vacuum and resuspend it in 12
ml of sterile H20 or T10E1. Store at –20°C.

g) Sodium Acetate: 3 M pH 5.2

Dissolve 2.46 g of CH3COONa (MW 82.03) in 10 ml total of H2O
and adjust the pH to 5.2 by adding CH3COOH. Sterilize in
autoclave.

h) Potassium Acetate: 7.5 M

Dissolve 5.78 g in 10 ml of distilled water and sterilize by
filtration.

i) Dextran Sulfate: 50%

Dissolve 50 g in distilled water; adjust the volume to 100 ml. Heat
in a water bath at 60–70°C to assist complete dissolution. Store
at –20°C.

j) Sodium Phosphate: 1M pH 6.5

Prepare: Sodium phosphate monobasic (Na H2PO4) 1 M,
dissolve 13.79 g in 100 ml of distilled water. The pH is approx.
4.2

Prepare: Sodium phosphate dibasic (Na2HPO4) 1M, dissolve

14.19 g in 100 ml of distilled water. The pH is approx. = 9.15. Mix
the two solutions 1:1 to obtain final pH = 6.5.

k) NaCl 5 M
Dissolve 29.2 g in 100 ml of distilled water and autoclave.

l) MgCl2 1 M:
Dissolve 20.3 g in 100 ml of distilled water and autoclave.

m) Tris-HCl 2 M pH 7.5
Dissolve 24.2 g in 80 ml of distilled water; adjust pH to 9.5 with
concentrated HCl. Complete volume to 100 ml and autoclave.

n) Tris-HCl 1 M pH 9.5
Dissolve 12.1 g in 80 ml of distilled water; adjust pH to 9.5 with
concentrated HCl. Complete volume to 100 ml and autoclave.

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 o) T10 E1
Dilute 5ml of solution m: Tris-HCl 2M pH 7.5 and 200 µl of
solution p: EDTA 0.5 M pH 8.0 in a total volume of 100 ml. Divide
in aliquots and autoclave. Store at –20°C.

p) EDTA 0.5 M pH 8.0

Dissolve 18.6 g EDTA in 50 ml of distilled water. Stir vigorously
on a magnetic stirrer. Adjust pH to 8.0 with pellets of NaOH.
Dispense into aliquots and sterilize by autoclaving. The disodium
salt of EDTA will not go into solution until the pH of the solution is
adjusted to approximately 8.0 by the addition of NaOH.

Solutions required:

• Labeling: kit (BRL), labeled nucleotide (BRL), SDS 5% (dil. stock

20%), Yeast t-RNA 20 mg/ml, NH4-AC 7.5 M, Ethanol absolute,
1XSSC (dil. 20XSSC and autoclave), Na-AC 3 M pH 5.2.

• Prehybridization and hybridization: Formamide, 20X SSC, Denhardt’s

solution 50X, Herring Sperm DNA 10 mg/ml, Na-phosphate 1 M pH
6.5, Dextran sulfate 50%.

• Washing: 20XSSC, SDS 20%, Tween-20.

• Blocking: Tris-HCl 2 M pH 7.5 NaCl 5 M, BSA, SA-AP, Tris-HCl 1 M

pHMgCl2 1 M, NBT, BCIP, n, n-Dimethylformamide.

Recommended Literature
Audy, P., J.G. Parent and A. Asselin. 1991. A note on four non-
radioactive labeling systems for dot hybridization detection of potato
viruses. Phytoprotection 72: 81– 86.
Hoppe, H.E.; L. Gliavedoni, M.A. Mandel, A. Arese, B. Orman, F. Bravo
Almonacid, H.N. Torres, and A.N. Mentaberry. 1988. Biotinylated
nucleic acid hybridization probes for potato virus detection. Archives
of Virology 103: 231– 241.
Owens, R.A. and T.O. Diener. 1981. Sensitive and rapid diagnosis of
potato spindle tuber viroid disease by nucleic acid hybridization.
Science 213: 670– 672.
Salazar, L.F., and M. Querci. 1992. Detection of viroids and viruses by
nucleic acid probes. In: Duncan, J.M. and L. Torres (eds.).
Techniques for the rapid detection of plant pathogens, pp. 129–144.
Blackwell Scientific Publications, Cambridge.

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